As a service to NHGRI investigators, the Embryonic Stem Cell and Transgenic Mouse Core specializes in generating genetically altered mouse lines for basic studies of gene function and regulation and for the creation of mouse models of human genetic diseases. Three separate technologies are utilized by the Core to generate genetically altered mice. The first method is to create conventional transgenics by microinjection of DNA into fertilized embryos (pronuclear microinjection) to generate germline mice. Secondly, targeted transgenics are generated by microinjecting genetically altered embryonic stem cells (ES cells). The ES cells are modified via homologous recombination of targeted genes in the Core or, are imported ES cell lines from other institutions (i.e., IKMC) Imported lines require expansion and archiving, DNA/RNA analysis, karyotyping, and MAP/Mycoplasma testing. ES cells are then injected into 2.5 day 8-cell embryos or 3.5 day blastocysts to generate fully ES cell-derived or chimeric mice, respectively. The Core generates transgenic mice by aggregation of tetraploid embryos with hybrid ES cells. By using 8-cell or tetraploid aggregation embryos to produce heterozygous or fully ES cell derived mice, the time and number of animals required is reduced for breeding chimeras. Third, the Core supports a genome-wide approach to discover and study new genes by ENU mutagenesis. Mutagenized mice are bred to assess stability of phenotype and heritability and then transferred to the investigator. The Core archives, in multiple locations, mutant strains by cryopreservation of sperm and embryos and reconstitutes the lines by in vitro fertilization. Thus, the Core can rapidly re-establish mouse strains in the event of a disaster or outbreak as well as easily export lines more efficiently and humanely to other institutions. For quality control, the Core cryopreserves stock embryos from wild-type strains (C57Bl/6J, FVB/N, 129S6Sv/Ev and Balb/c). These embryos are also used for flexible microinjection of 8-cell embryos. The use of cryopreserved embryos reduces animal donor needs by 50-60% and allows microinjection of 2-3x more embryos per session. An additional service provided to our institute is to rederive animals into our facility by embryo transfer of fertilized eggs. Rederivation generally takes 7-8 weeks as compared to 14-16 weeks for conventional quarantine methods. Closely associated to our rederivation and cryopreservation program, is the in-house breeding colony. This colony rapidly generates mice for experiments and centralizes animals used across animal protocols (ie. cre transgenics). We perform PCR-based genotyping, breed on multiple backgrounds (129 and C57Bl/6) and cryopreserve the lines. We can readily reconstitute mice, which enables reduced rack space for maintenance breeding. Other services include embryo dissection, mouse perfusions, injections, colony maintenance, and animal identification by genotyping. The Core works with NHGRI investigators in construct design, and in basic manipulations of mouse husbandry. The Core is committed to cutting edge transgenic technologies while finding better avenues to reduce the animal requirements. For example, we are investigating inhibitors to improve ES cell quality and improved chimeras. By using inhibitors of GSK3b and MAPK, our parental cell lines and targeted lines have better morphology, controlled growth rate, and higher Nanog levels. This should improve both in-house, and the multiple imported ES cells. The Core is also standardizing protocols for creating induced Pluripotent Stem (iPS) cells from mutant mice. We are using dox-inducible lentiviruses containing the inductive factors and mRNA reprogramming. Our objectives are to generate iPS cells from transgenic mouse embryonic feeder cells (MEFS) or tail tip fibroblasts (TTF), characterize them by immunocytochemistry, real-time PCR and generate chimeras from these ES-like cells and breed to germline transmission. We prepare MEFS or tail tip fibroblasts and make them available to our investigators who require them for human ES and iPS studies. Moreover, we support characterization of human iPS lines by assisting in the cell injection and generation of teratomas in nude mice. The Core is developing protocols for genome editing using TALENs and Crispr/Cas systems. The site specific nuclease systems will allow production of genetically engineered mice with multiple gene modifications in a one step process. Core Personnel Description and Equipment Capabilities: The Transgenic Core has seven full-time employees. Lisa Garrett directs and oversees daily operations, training and experimental design. The core staff includes six staff, employed directly by the core, through a branch, or by contract. They include Jun Cheng, Gene Elliott, Kowser Hasneen, Karen Hazzard (all technical staff) and Cecilia Rivas and Elsa Escobar (animal support contractors). The physical organization of the Core is divided into two laboratories behind the animal barrier. The third floor lab houses the central tissue culture space for ES cell growth and maintenance, a cryopreservation area, a microinjection suite with three Zeiss/Eppendorf microinjection stations and one Nikon micromanipulation station, a molecular biology area, and administrative space for 5 individuals. The second floor laboratory is for animal use with downdraft tables and biosafety cabinets. We have a dedicated area for harvesting embryos and tissues with 4 stereomicroscopes that are available to institute investigators. This lab contains a chemical fume hood for ENU, a small animal surgery suite with 3 stereomicroscopes and downdraft tables, a tissue culture area for iPS generation, and ES cell differentiation. There is administrative space for 2 individuals. Summary June 2012-2013 The Core generated conventional transgenics from 8 DNA constructs since June 2012. We have 32 ES cell - targeting constructs that are in various stages of development such as screening for homologous recombination, microinjection, and generation of germline transmitting progeny. I anticipate greater than 20 conventional transgenic constructs for the upcoming year and at least 35 targeting constructs for our institute. During the past year, we rederived in 22 lines of imported mice, cryopreserved 45 lines for disaster preparation, and archived 83 mouse lines. For the upcoming year, we will continue to cryopreserve all mutant mice imported or generated by the Core. I have modified our freezing program to reduce the numbers of embryos frozen (therefore reducing the numbers of mice) and balance the archiving with cryopreserved sperm. We have a significant increase in efficiency of recovering frozen sperm by IVF (>50%) using a modified method by Nakagata et al., J.Mamm.Ova Res. 2010. The ENU mutagenesis project has been successful with 12 mutants identified from the screening of 811 male G1 progeny. From the inception of the ENU program, approximately 1,675 G1 male progeny have been produced. The present objective for this screen is to continue generating mutagenized G1 males that will be bred to sensitized animals for neural crest phenotypes until the gene pool has been saturated and redundant mutations appear. From reviewing Pubmed and information from our investigators, the Core has made substantial contributions on >120 papers from 2005-present. This includes both co-authorship and acknowledgements of several members of the Core. This compilation of papers represents any resource or mouse generated by the Core. Since January 2011, the Core has had co-authorship on at least 5 publications.